The cytochrome P450 family of enzymes is primarily responsible for the metabolism of xenobiotics such as drugs, carcinogens and environmental chemicals, as well as several classes of endobiotics such as steroids and prostaglandins. Members of the cytochrome P450 family are present in varying levels and their expression and activities are controlled by variables such as chemical environment, sex, developmental stage, nutrition and age.
More than 200 cytochrome P450 genes have been identified. There are multiple forms of these P450 and each of the individual forms exhibit degrees of specificity towards individual chemicals in the above classes of compounds. In some cases, a substrate, whether it be drug or carcinogen, is metabolized by more then one of the cytochromes P450. Genetic polymorphisms of cytochromes P450 result in phenotypically-distinct subpopulations that differ in their ability to perform biotransformations of particular drugs and other chemical compounds.
These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to most humans may cause toxic side-effects in an individual suffering from a defect in an enzyme required for detoxification of the drug. Alternatively, a drug that is effective in most humans may be ineffective in a particular subpopulation because of lack of a enzyme required for conversion of the drug to a metabolically active form. Further, individuals lacking a biotransformation enzyme are often susceptible to cancers from environmental chemicals due to inability to detoxify the chemicals. Eichelbaum et al., Toxicology Letters 64165, 155-122 (1992). Accordingly, it is important to identify individuals who are deficient in a particular P450 enzyme, so that drugs known or suspected of being metabolized by the enzyme are not used, or used only with special precautions (e.g., reduced dosage, close monitoring) in such individuals. Identification of such individuals may indicate that such individuals be monitored for the onset of cancers.
Cytochrome P450 2D6, also known as debrisoquine hydroxylase, is the best characterized polymorphic P450 in the human population (Gonzalez et al., Nature 331:442-446 (1988)). A poor metabolizer phenotype has been reported which behaves as an autosomat recessive trait with an incidence between 5 and 10% in the white population of North America and Europe. Poor metabolizers exhibit negligible amounts of cytochrome P450 2D6 (Gonzales et al., supra). Genetic differences in cytochrome P450 2D6 may be associated with increased risk of developing environmental and occupational based diseases. See Gonzalez & Gelboin, J. Toxicology and Environmental Health 40, 289-308 (1993)).
Several drugs for treating cardiovascular and psychiatric disorders are known substrates of cytochrome P450 2D6. (Dahl and Bertilsson, Pharmacogenetics 3, 61-70 (1993)), a situation that creates problems in prescribing such drugs. Although such drugs may be the most effective treatment for most of the population, physicians are reluctant to prescribe them due to the risk of adverse effects in poor metabolizers. Buchert et al., Pharmacogenetics 2, 2-11 (1992); Dahl et al., Pharmacogenetics 3, 61-70 (1993).
Existing methods of identifying deficiencies in patients are not entirely satisfactory. Patient metabolic profiles are currently assessed with a bioassay after a probe drug administration. For example, a poor drug metabolizer with a 2D6 defect is identified by administering one of the probe drugs, debrisoquine, sparteine or dextromethorphan, then testing urine for the ratio of unmodified to modified drug. Poor metabolizers (PM) exhibit physiologic accumulation of unmodified drug and have a high metabolic ratio of probe drug to metabolite. This bioassay has a number of limitations: lack of patient cooperation, adverse reactions to probe drugs, and inaccuracy due to coadministration of other pharmacological agents or disease effects. See, e.g., Gonzalez et al., Clin. Pharmacokin. 26, 59-70 (1994). Genetic assays by RFLP (restriction fragment length polymorphism), ASO PCR (allele specific oligonucleotide hybridization to PCR products or PCR using mutant/wildtype specific oligo primers), SSCP (single stranded conformation polymorphism) and TGGE/DGGE (temperature or denaturing gradient gel electrophoresis), MDE (mutation detection electrophoresis) are time-consuming, technically demanding and limited in the number of gene mutation sites that can be tested at one time.
A serious complication in patient drug choice is that most drugs have not been characterized for their metabolism by P450 2D6 and other cytochromes P450. Without knowing which cytochrome(s) p450 is/are responsible for metabolizing an individual drug, an assessment cannot be made for the adequacy of a patient's P450 profile. For such drugs, there is a risk of adverse effects if the drugs are administered to poor metabolizers.
Possible roles of monoclonal antibodies in cytochrome P450 research including detection, immunolocalization, immunopurification and phenotyping of particular cytochrome P450 isozymes are reviewed by Gelboin, Pharmacological Reviews 45, 413-453 (1993). Cribb et al., Drug Metabolism & Disposition 23, 671-675 (1995) discuss polyclonal antibodies raised against two peptides that correspond to a 33 amino acid sequence of cytochrome P450 2D6. They report that antisera raised against a peptide corresponding to amino acids 254-273 inhibited up to 95% of dextromethorphan 0demethylase activity in human liver microsomes but lacked crossreactivity with recombinant cytochromes P450 1A2, 2C9, 2E1, or 3A4. Gonzalez et al., (1988), supra report that a rat polyclonal antibody to P450 2D6 enzyme inhibited human liver microsomal bufuralol F-hydroxylase, and human purified 2D6 protein in an in vitro reconstituted enzyme assay. Prueksaritanont et al., Biochemical Pharmacology 50, 1521-1525 (1995) report a polyclonal P450 2D6 antibody raised against a 20 amino acid peptide containing residues 243-273 of the native protein. The polyclonal antibody was used to compare (+)-bufuralol F-hydroxylation in human and rhesus monkey intestinal microsomes with hepatic microsomes. It was reported that the polyclonal anti-cytochrome P450 2D6 antibody inhibited bufuralol metabolism in human liver by about 75%. Certain mouse monoclonal antibodies to human cytochrome P450 2D6 are discussed by Transon et al., Eur. J. Clin. Pharmacol. 51, 79-85 (1996) and Willis et al., Biochemical Pharmacol., 44, 617-620 (1992). Transon et al. note that different antibodies to 2D6 vary greatly in properties. None of the monoclonals discussed by Transon et al. or Willis et al. are reported to inhibit 2D6 activity.
The present invention provides inter alia monoclonal antibodies that specifically bind to human cytochrome P450 2D6 and inhibit its activity. The monoclonal antibodies can be used to screen drugs for their metabolism by 2D6 and/or identify 2D6 poor metabolizers by simple bioassays, thereby overcoming the problems in prior complicated methods discussed above.